The process of blood coagulation involves a series of proteins known as blood coagulation proteins which act in a cascade fashion to effect the formation of a blood clot. Hemophilia is a disease of humans and other mammals wherein a gene encoding a blood coagulation factor contains a mutation such that the encoded protein does not function normally in the cascade process. Specifically, the hereditary disease, hemophilia B, is characterized by a mutation in the gene encoding the blood coagulation protein, Factor IX (F.IX). F.IX is reviewed in High et al. (1995, "Factor IX" In: Molecular Basis of Thrombosis and Hemostasis, High and Roberts, eds., Marcel Dekker, Inc.).
Adenoviral vectors are well known in gene therapy and have been used to effect expression of high levels of canine factor IX in immunodeficient mice or in immunocompetent mice when the virus is administered in conjunction with immunosuppressive agents. When adenoviral vectors are administered to immunocompetent mice in the absence of immunosuppressive agents, these vectors induce a strong inflammatory and cytotoxic T lymphocyte (CTL) response (Dai et al., 1995, Proc. Natl. Acad. Sci. USA 92:1401-1405) which negates the beneficial effects of the therapy. In addition, there are reports which suggest that intramuscular injection of replication defective adenovirus provides long-term expression of a transgene, provided that the transgene encodes a self-protein (i.e., a host protein), such that a strong host immune response is avoided (Tripathy et al., 1996, Nature Med. 2:545-550; Yang et al., 1996, Hum. Mol. Genet. 5:1703-1712). Thus, while there has been significant progress in the area of gene therapy in in vivo expression of a selected transgene following direct injection of an adenoviral vector into skeletal muscle, the use of adenoviral vectors may not be the optimal method for gene therapy in light of these immunological considerations.
Retroviral vectors have also been used experimentally as a model for treatment of hemophilia B. However, levels of expression of F.IX from these vectors are reported to be too low to be of therapeutic value (Kay et al., 1993, Science 262:117-119).
Plasmid DNA which has been injected into mouse muscle has been shown to direct expression of erythropoietin (Epo) (Tripathy et al., 1996, Proc. Natl. Acad. Sci. USA 93:10876-10880), but this method of gene therapy is apparently not sufficiently efficient for the expression of a gene product such as F.IX, which is needed at relatively high levels in the circulation (compared with Epo) to achieve a therapeutic effect.
Adeno-associated virus (AAV) is an alternative vehicle to adenovirus for delivery of genes to muscle. Recombinant AAV (rAAV) does not contain sequences encoding viral proteins and has the potential to integrate into the chromosomal DNA of the host cell (Carter, 1992, Curr. Opin. Biotech. 3:533-539; Skulimowski et al., 1995, Method Mol. Genet. 7:7-12). Production and purification procedures are now available which facilitate the generation of pure rAAV which is not significantly contaminated by wild-type AAV or helper adenovirus (Skulimowski et al., 1995, supra; Fisher et al., 1996, J. Virol. 70:520-532; Samulski et al., 1989, J. Virol. 63:3822-3828). As noted herein, administration of adenovirus to mammals is accompanied by the aforementioned immunological problems.
While the efficiency of in vivo transduction with rAAV in the absence of helper virus is low for hepatocytes and airway epithelial cells (Fisher, 1996, supra), certain post-mitotic cells such as neurons (Kaplitt et al., 1994, Nature Genet. 8:148-154) and skeletal muscle fibers (Xiao et al., J. Virol. 70:8098-8108) can be effectively transduced with this vector. Stable expression of lacZ for up to 1.5 years has been reported (Xiao et al., supra). In contrast to adenoviral vectors, intramuscular injection with rAAV in immunocompetent animals does not result in a CTL response against transduced muscle fibers, nor are circulating antibodies against the intracellular lacZ gene product present.
The expression of the secreted protein, Epo, following intramuscular injection with rAAV is reported in Kessler et al. (1996, Proc. Natl. Acad. Sci. USA 93:14082-14087). However, the levels of protein expression reported were one to two orders of magnitude below that required for a therapeutic effect mediated by F.IX.
Current therapy for hemophilia involves the intravenous injection of a preparation of clotting factor concentrates whenever a bleed occurs. This treatment is cumbersome, inconvenient and very expensive. The average patient pays approximately $100,000 per year for the concentrate alone. Further, because the concentrate is only administered to the patient intermittently, patients remain at risk for life-threatening bleeds which are fatal if treatment is not timely administered.
There is a long felt and acute need for methods of delivering F.IX to mammals having hemophilia, in particular, to humans having hemophilia, such that a therapeutic effect is achieved. The present invention satisfies this need.